DNA polymerase enzymes are naturally-occurring intracellular enzymes, and are used by a cell to replicate a nucleic acid strand using a template molecule to manufacture a complementary nucleic acid strand. The in-vitro use of enzymes having DNA polymerase activity has in recent years become more common in a variety of biochemical applications including cDNA synthesis and DNA sequencing reactions (see Sambrook et al., (2nd ed. Cold Spring Harbor Laboratory Press, 1989) hereby incorporated by reference herein), and amplification of nucleic acids by methods such as the polymerase chain reaction (PCR) (Mullis et al., U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159, hereby incorporated by reference herein) and RNA transcription-mediated amplification methods (e.g., Kacian et al., PCT Publication No. WO91/01384 which enjoys common ownership with the present application and is hereby incorporated by reference herein). Methods such as PCR make use of cycles of primer extension through the use of a DNA polymerase activity, followed by thermal denaturation of the resulting double-stranded nucleic acid in order to provide a new template for another round of primer annealing and extension.
Various scientific and industrial applications exist in which it would be advantageous to use a DNA polymerase that function efficiently at high salt concentrations. In sequencing, GC compressions can be resolved by using high salt concentrations. In nanopore sequencing high salt concentration boosts the signal to noise ratio for ionic-current-based nanopore measurements. Salt tolerant DNA polymerases may be found among members of the extreme halophiles, in which salt tolerance is achieved not by exclusion of monovalent ions from the cytosol, but by adapting intracellular machinery function in elevated salt. As an example of salt tolerance among members of the extreme halophiles, malate dehydrogenase from the archaeal halophile Haloarcula marismortui incorporates a salt-adaptive strategy where the high ionic concentration from the environment is not only tolerated but is incorporated within the protein. Sodium and chloride ions are found incorporated within the molecule itself. When considering viruses that infect extreme halophiles, not only are proteins of the viral capsid exposed directly to the environment, but the proteins of the replication machinery must operate effectively within the elevated salt environment of its archaeal host.
Phi29 is a widely used and commercially successful salt tolerant DNA polymerase from bacillus phage Phi29, however its salt tolerance is limited, and in 1 M KCl there is no detectable binding of the enzyme to the DNA substrate.
It would be desirable to identify DNA polymerases that can function at elevated salt concentrations, for example at concentrations of at least 5%, 10%, 20% or 30% wt/vol salt, such as KCl or NaCl.